Single-cell assessment of trophoblast stem cell-based organoids as human placenta-modeling platforms

The recent discovery of human trophoblast stem cells (hTSC) and techniques allowing for trophoblast organoid (TOrg) culture have established promising approaches for studying human trophoblast development. To validate the accuracy of these models at single-cell resolution, we directly compared in vitro TOrg cultures derived from primary progenitor cytotrophoblasts (CTB) or commercially available hTSC lines to in vivo human trophoblasts using a scRNA-seq approach. While patient-derived (PD)- and hTSC-derived TOrgs overall reflect cell differentiation trajectories with accuracy, specific features related to trophoblast state make-up, distinct sub-paths of differentiation, and predicted transcriptional drivers regulating stem cell maintenance were shown to be misaligned in the in vitro platforms. This is best exemplified by the identification of a distinct progenitor state in hTSC-derived TOrgs that showed characteristics of CTB- and extravillous-like cell states. Together, this work provides a comprehensive resource that identifies underlying strengths and limitations of current TOrg platforms. Summary Statement Single-cell transcriptomics provides comprehensive comparison between trophoblast organoids derived from commercially available trophoblast stem cells and first-trimester primary human cytotrophoblasts. HIGHLIGHTS IN BRIEF While the merits and utility of current trophoblast organoid cultures have been established, high-resolution assessment and comparison of conserved and divergent features of these systems to cell states and differentiation trajectories of trophoblasts in situ or in vitro has not been performed. Here, Shannon et al. generate a single-cell transcriptomic atlas of two trophoblast organoids that comprehensively define the similarities and discrepancies in relation to trophoblasts from the placental-maternal interface. ### Competing Interest Statement The authors have declared no competing interest. * cCTB : Column cytotrophoblast CTB : Cytotrophoblast DGE : Differential gene expression EVT : Extravillous trophoblast GA : Gestational age HLA : Human leukocyte antigen hTSC-TOrg : Human trophoblast stem cell line-derived trophoblast organoid MPI : Maternal-Placental interface ( in vivo ) trophoblasts PD-TOrg : Patient-derived trophoblast organoid scRNA-seq : single-cell RNA sequencing SCT : Syncytiotrophoblast SCTp : Syncytiotrophoblast precursor TOrg : 3D trophoblast organoid UMAP : Uniform manifold approximation and projection